Species of Bacillus, including B. anthracis and B. cereus, present significant health hazards. B. anthracis is the highly pathogenic, gram-positive bacteria that is responsible for the acute and often fatal disease, anthrax. It is categorized as a Type A pathogen by the National Institute of Allergy and Infectious Disease. Current therapeutics for B. anthracis infections have serious limitations including expense, resistance and contraindications in children. B. anthracis and B. cereus are closely related, in fact, biochemical and genetic evidence suggests that they should be considered a single species. Exposure to B. cereus, usually as a food contaminant, causes a number of infections including endophthalmitis, bacteremia, septicemia, endocarditis, pneumonia and meningitis, some of which have proven fatal.
Cryptosporidium and Toxoplasma are apicomplexan parasitic protozoa that cause severe disease in the population worldwide. Cryptosporidiosis, caused by C. hominis, is characterized by wasting disease and most often affects immune-compromised patients, the elderly, and day-care children, although very large outbreaks have occurred in otherwise healthy populations. Effective therapy for cryptosporidiosis is hard to find. At this time, there is a single approved therapeutic agent, nitazoxanide, against Cryptosporidium although the application of this drug is limited to immune-competent patients and effects have not been studied in children under 12 years of age. Toxoplasmosis, when transmitted congenitally as T. gondii, can cause neonatal death and, when transmitted through ingestion of contaminated meat or water, can cause fever and sore throat, or cerebral inflammation in immune-compromised patients. Both of these parasitic protozoa have been classified as Category B biodefense agents.
Systemic fungal infections are a significant and increasing cause of death and severe illness worldwide. Mortality rates due to Candida spp. infections were 38% between 1983 and 1986 and 49% between 1997 and 2001. The incidence of these infections has risen because of the increased number of immune-compromised patients. Up until the 1980s, Candida albicans was the primary cause of systemic candidemia infection and could be treated with traditional therapeutics including azole derivatives and amphotericin B. However, shifting epidemiology dictates that while C. albicans infections still represent the majority (˜50%), other species of Candida, primarily C. glabrata, now cause a significant (˜20%) number of bloodstream infections.
Staphylococcus aureus is a facultative anaerobic gram-positive coccal bacterium that is a major cause of hospital-acquired infections, most frequently associated with the bloodstream, skin and soft tissue, ventilator-assisted pneumonia and catheters. The increasing frequency of infections caused by methicillin-resistant S. aureus (MRSA) is of particular concern. Community-acquired MRSA (CA-MRSA), genotypically distinct from HA-MRSA, has also now become an established threat among patients without traditional risk factors. In addition to S. aureus, the Gram-positive bacteria Streptococcus pyogenes is a major cause of complicated skin and skin structure infections (SSTI). Reliably distinguishing between infections caused by S. aureus and S. pyogenes is difficult because of overlaps in clinical presentation.
Dihydrofolate reductase (DHFR) has been a validated drug target for the treatment of bacterial and protozoal infections for decades. DHFR is an essential enzyme and plays a key role in the folate biosynthetic pathway. Occurring as a bifunctional protein with thymidylate synthase (DHFR-TS) in the apicomplexan protozoa, DHFR utilizes the cofactor NADPH to catalyze the reduction of dihydrofolate to tetrahydrofolate, thereby performing a key reaction in the sole de novo synthesis of deoxythymidine monophosphate (dTMP). Since DHFR is an essential enzyme to all cells, inhibitors targeting pathogenic organisms must be selective as well as potent in order to avoid complications resulting from inhibiting the human enzyme. The difficulty of achieving both potency and selectivity for Cryptosporidium DHFR was underscored by a study from Nelson and Rosowsky (Antimicrob Agents Chemother (2001) Dec; 45(12): 3293-303) in which they examined 96 structurally diverse DHFR inhibitors and were unable to identify compounds that were both potent and selective for C. hominis DHFR. DHFR has been widely conserved throughout evolution. However, several residue differences exist in the active sites of different species that make achieving selectivity for the pathogenic form of the enzyme possible.
The DHFR inhibitor pyrimethamine (i.e., 5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine) has been effectively used to treat toxoplasmosis as well as malaria, caused by another apicomplexan parasite, Plasmodium (such as against Plasmodium falciprum). However, many patients have had severe reactions to pyrimethamine, limiting its efficacy.
Trimethoprim (i.e., 5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4-diamine) has been used effectively in the clinic as an antibacterial agent since the 1960s. For example, it has been used against E. coli and species of Streptococcus. It possesses excellent drug-like characteristics including a relatively low molecular weight (MW=290 Daltons). However, trimethoprim also exhibits a high affinity for only a small subset of species of DHFR from pathogenic organisms such as Escherichia coli. This limits its widespread application. Moreover, trimethoprim exhibits only moderate in vitro potency against DHFR from C. hominis (ChDHFR) and T. gondii (TgDHFR). Trimethoprim is less potent against DHFR from Cryptosporidium and Toxoplasma, two Apicomplexan protozoa.
Amphotericin B and azole derivatives have traditionally been used to treat C. albicans infections. However, other species of Candida, primarily C. glabrata have a lower susceptibility toward azole compounds, especially the commonly used agent, fluconazole. The therapeutic window to treat C. glabrata is even narrower since C. glabrata strains are also often resistant to Amphotericin B.
While vancomycin is the preferred treatment for MRSA infection in hospitals, vancomycin-intermediate S. aureus isolates (VISA) and vancomycin-resistant S. aureus (VRSA) strains have been reported in the US since 2002. Additionally, many strains of S. aureus, including new strains of CA-MRSA, show sensitivity to trimethoprim-sulfamethoxazole (TMP-SMZ). Resistance to TMP-SMZ among staphylococci has been observed in Australia and the United States since the early 1980s. Surveys from a collection of strains show that 28% of MRSA isolates are TMP-resistant and 35% are SMZ-resistant. Likewise, the spectrum of agents that may be effective against both S. aureus and S. pyogenes is limited by resistance: S. pyogenes can be resistant to macrolides and S. aureus is frequently resistant to beta-lactam antibiotics. Therapeutics with activity against MRSA and S. pyogenes would be ideal agents for treating SSTI. Thus, the development of an efficacious antibacterial agent that targets both organisms is a high priority.
During drug development, it is often appreciated that during the lead optimization process, increases in potency correlate with increases in molecular weight. However, the additional molecular weight frequently represents a liability as it compromises the drug-like properties of the lead. The necessary compromise between increasing molecular weight and increasing affinity can be examined quantitatively with the concept of ligand efficiency. Ligand efficiency is defined as the overall binding energy per nonhydrogen atom; a higher ligand efficiency defines a superior compound. Interestingly, although methotrexate (i.e., (S)-2-(4-(((2,4-diaminopteridin-6-yl)methyl)methylamino)benzamido)pentanedioic acid or MTX) is a much more potent compound (IC50 values against ChDHFR and TgDHFR are 23 nM and 14 nM, respectively) than trimethoprim, it has the same ligand efficiency as trimethoprim, implying that the increased potency is largely dependent on the increased molecular weight rather than a more optimal positioning of pharmacophoric elements.
Therefore, what are needed are pharmaceutical compositions containing relatively low molecular weight compounds that are specific and effective for the treatment of various pathogenic infections.